14 September 2009
Inability to generate NOD ES cells capable of robust chimerism and germline transmission has been a longtime frustrationto researchers using this key model. Definition of the ground-state culture requirements for sustaining ES cell pluripotency has enabled generation of ES cell lines from mouse strains previously found to be recalcitrant and also from rats. We have applied this approach which uses small-molecules to inhibit differentiation together with LIF to generate multiple lines of NOD ES cells. These lines are stable, retaining normal karyotypes and expression of pluripotency markers, until at least passage 14. We injected several of the male lines into C57BL/6 blastocysts, and all generated chimeras. The majority of the chimeric mice were male, indicating efficient sex conversion of the host blastocyst. Most of the chimeric mice showed extensive contribution from the injected ES cells, as detected by coat color, the extent of hematopoietic chimerism and the presence of NOD polymorphic markers. Notably, we detected islet infiltrates in several of the male chimeric mice, and, indeed, two spontaneously developed type 1 diabetes. Importantly, our data furthermore show that these ES cells are clearly capable of robust germline transmission. Female germline offspring from these chimeras spontaneously developed type 1 diabetes at a tempo and incidence characteristic of the NOD mouse strain (Figure 1a). Pancreatic infiltration seen in these mice results in the beta cell destruction leading to diabetes (Figure 1b, T cells are marked up in green and insulin producing beta cells in red.).
One of the major reasons that cells such as these are desirable is that they provide the means to identify definitivelyalleles of genes contributing to diabetes development. Genetic manipulation via NOD ES cells will enable geneticists to determine whether candidate gene loci identified by mapping studies are indeed the genes involved in predisposing to diabetes development or whether other nearby genes in linkage disequilibrium are responsible. We have shown that our NOD ES cells can be genetically modified and still retain their capacity to contribute to the germline. These results indicate that our NOD ES cells will indeed provide a means of carrying out detailed genetic analyses.
These cells can be differentiated in vitro, and it will be crucial to establish whether they can be differentiated into beta cells capable of sustained insulin production and glucose responsiveness that can be retained in vivo following transplantation. Such studies will enable researchers to determine whether NOD ES cells can be used to provide a source of replacement beta cells and assess the efficacy of tolerogenic strategies to prevent their autoimmune destruction. They will also allow investigators to assess whether there is any genetic factor influencing beta cell development in NOD mice that may render their beta cells more susceptible to autoimmune destruction and diabetes development.
In summary, we have applied ground-state culture conditions to derive NOD ES cells that should prove useful for a range of studies that will enable the diabetes research community to dissect the processes leading to development of type 1 diabetes.
Read more in: